The present invention relates to a method for the preparation of a fungal body and a lipid rich in .gamma.-linolenic acid therefrom. More particularly, the invention relates to a method for the preparation of a fungal body of certain filamentous fungi belonging to the genus of Mortierella containing a large amount of lipids by cultivation under specific conditions and a lipid therefrom which is rich in the content of .gamma.-linolenic acid.
It has been hitherto reported that several fungal bodies contain a considerably large amount of lipids including neutral lipids, i.e. oils and fats, and polar lipids, i.e. phospholipids and glycolipids, in a content of, for example, 25 to 65% by weight on the dry basis. Such high-lipid filamentous fungus moulds are exemplified by Geotricum candidum, Fusarium lini, Fusarium buligenum, Penicillium lilacinum, Penicillium soppi, Penicillium spinulosum, Aspergillus nidulance and Mucor sacineleudes reported by K. Yamada [Food-Industrial Microbiology, page 63], Mortierella vinacea reported by C. G. C. Chester et al. [J. Gen. Microbiol., volume 41 (1965), page 127] and Aspergillus terreus, Aspergillus ochraceus, Cladosporium fulvum, Cladosporium herbarum and Penicillium gladioli reported by J. Singh et al. [J. Sci. Fd. Agric., volume 23 (1972), page 1113].
These fungi can be cultured in a culture medium containing a carbohydrate as the carbon source and the preferable concentration of the carbohydrate as the nutrient in the culture medium is reportedly 20 to 60 g/liter when they are cultured in a flask or in a small-size culture tank. When a fungal body of high lipid content is desired of these fungi, the velocity and extent of the multiplication of the filamentous fungal body are usually not so high that the carbohydrate as the carbon source is not exhaustively consumed. For example, it is reported by C. G. C. Chester et al. that the best results obtained in the culture of Mortierella vinacea were that the use of a culture medium containing glucose in an initial concentration of 21.2 g/liter at 20.degree. C. gave 4.7 g/liter of the fungal body as dried containing 3 g/liter of the lipid extractabale therefrom after consumption of 17 g/liter of starting glucose for 10 days. When molasses is used as the carbon source in the culture of Penicillium spinurosum, it is noted that the increase in the concentration of the carbon source in the culture medium does not contribute to the increase of the multiplication of the fungal body so much with rather decreased proportion of the consumed carbon source on the contrary. In this case, the highest results reported by A. W. Khen et al. [Can. J. Microbiol., volume 7 (1961), page 895] are that the amount of the fungal body multiplication as dried was 22 g/liter corresponding to only 2.5 g/liter of the lipid extractable therefrom after 6 days of culture at 30.degree. C. starting with a molasses concentration of 164 g/liter of which about 40% is consumed by the multiplication of the fungus.
Turning now to the nature or constituents of the lipids derived from such a fungal body, a recent concern in the pharmaceutical industry is the preparation of .gamma.-linolenic acid from the lipids. As is well known, .gamma.-linolenic acid, i.e. cis-6,9,12-octadecatrienoic acid which cannot be synthesized in the living body, is one of the essential fatty acids indispensable in the diet for mammals including human. This is because .gamma.-linolenic acid taken into a living body is a precursor of and converted into bis-homo-.gamma.-linolenic acid and further into arachidonic acid which in turn are converted into prostaglandins E.sub.1 and F.sub.1.alpha. and prostaglandins E.sub.2 and F.sub.2.alpha., respectively, playing very important roles in the living body so that deficiency of .gamma.-linolenic acid in the diet causes various diseases and disorder of the body requiring administration thereof in the form of a medicine.
.gamma.-Linolenic acid or a lipid containing the same is conventionally obtained by extracting from the seeds of several plants such as evening primrose (Oenothera biennis L.) but the productivity thereof is quite unsatisfactory. Therefore, great efforts are continuously directed, for example, by R. B. Wolt et al. to seek a plant giving seeds containing an oil rich in the content of .gamma.-linolenic acid [J. Amer. Oil Chem. Soc., volume 60 (1983), page 1858]. Unfortunately, all of the plants reportedly producing a lipid containing a promising amount of .gamma.-linolenic acid are rare with low availability so that collection of a large amount of the seeds thereof is a rather difficult matter with no possibility of industrial production of .gamma.-linolenic acid with high productivity.
An alternative possibility for the industrial preparation of .gamma.-linolenic acid or a lipid rich in the content thereof would be given by a microbiological method when and if a microorganism efficiently producing such a lipid is discovered and an industrially efficient method for the cultivation of such a microorganism is established since the microbiological production can be performed usually without supply of the sunlight energy so that the productivity is never affected by the weather. Any large facilities for the industrial production of .gamma.-linolenic acid can be constructed on a relatively small land area in comparison with the agricultural fields necessary for the growth of the specific plant and the rate of production can freely be controlled according to desire.
Several microorganisms are known to produce lipids containing a relatively large amount of .gamma.-linolenic acid including Mucor globosus and Mucor pusillus reported by R. O. Mumma et al. [Lipids, volume 6 (1971), page 584], Choanephora cucurbitarum reported by H. B. White, Jr. et al. [Biochim. Biophys. Acta, volume 116 (1966), page 388], Pythium debaryanum, Saprolegnia litoralis, Rhizopus stolonifer, Rhizopus arrhizus, Phycomyces blakesleeanus, Mucor javanicus and Helicostylum pyriforme reported by R. Shaw [Biochim. Biophys. Acta, volume 98 (1965), page 230] and Entomophtora coronata reported by R. O. Mumma et al. [Lipids, volume 5 (1970), page 915].
Each of the research works reported by the above named authors, however, is only experimental using a glass flask or a small culture tank and the starting concentration of the carbohydrate as the carbon source in the culture medium is limited to 20 to 60 g/liter. The amount of the fungal body obtained by the culture per unit volume of the medium is relatively small and the content of lipids in the fungal body is only 3 to 30% by weight so that the microbiological way so far developed is also not quite promising as an industrial method for the production of .gamma.-linolenic acid or a lipid rich in the content thereof insofar as the above named microorganisms are utilized as the objective of the culture.